Cytotoxic ribonuclease variants

ABSTRACT

This invention relates to cytotoxic variants of human ribonuclease 1 (RNase 1) identified through analysis of the interaction between RNase 1 and the human ribonuclease inhibitor (hRI) as defined by the three dimensional (3-D) atomic structure of the RNase1 hRI complex. Also disclosed is the 3-D structure of the hRI•RNase 1 complex and methods for designing the RNase 1 variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Nonprovisional applicationSer. No. 11/454,418 filed Jun. 16, 2006 now U.S. Pat. No. 7,655,757,which claims priority to U.S. Provisional Application No. 60/691,311filed Jun. 16, 2005. Each of these applications is incorporated byreference here in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with United States government support awarded bythe following agency: NIH CA073808 and GM 064598. The United Statesgovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

Ribonucleases are enzymes that catalyze the degradation of RNA. A wellstudied ribonuclease is bovine pancreatic ribonuclease A (RNase A), theputative biological function of which is to break down the large amountof RNA that accumulates in the ruminant gut. The RNase A superfamily isa group of RNase enzymes classified as similar to RNase A which possessa number of interesting biological properties includingantiproliferative, cytotoxic, embryotoxic, aspermatogenic, andantitumoral activities. One member of this family is a homolog of RNaseA, originally isolated from oocytes and early embryos of the Northernleopard frog Rana pipiens.

The frog (Rana pipiens) ribonuclease, when placed in a human cell, isnot strongly-inhibited by RI and its RNase activity destroys cellularRNA and kills the target cell. The anti-tumor properties, both in vitroand in vivo, of the frog ribonuclease are described and claimed in U.S.Pat. No. 5,559,212. This ribonuclease molecule is now known as Onconase®(ONC). The property of degrading RNA is essential to the cytotoxicity ofONC. ONC is currently being evaluated as a cancer therapeutic inclinical trials.

A significant limitation on the suitability of ONC as a chemotherapeuticis dose-limiting renal toxicity. ONC is retained in the kidney atconcentrations much greater than mammalian members of the RNasesuperfamily. There may also be allergenic issues with ONC, since miceproduce antibodies against ONC but not against RNase A, with which ONCshares about 30% of its amino acids. This suggests that other members ofthe RNase family may also be suitable candidates for evaluation asclinical therapeutics if they can be imbued with the cytotoxicproperties similar to ONC.

In mammals, levels of RNase activity are controlled by a ribonucleaseinhibitor (RI), which is a 50-kDa protein found in the cytosol of allmammalian cells. RI is a member of a leucine rich family of proteins andis composed of 15 alternating repeats arranged symmetrically in ahorseshoe shaped molecule. RI has a large number of cysteine residues(32 in human RI) which means that it can only keep its shape andfunction in a reducing environment like the cytosol. RI acts to bind tomembers of the RNase superfamily, one RI to one molecule of RNase, andwhen so bound, RI completely inhibits the catalytic activity of theribonuclease by steric blockage of the active site of the enzyme. Thebinding of RI to RNase is a very tight one, having a very high bindingaffinity.

Some RNase superfamily members, notably ONC and bovine seminalribonuclease, possess the native ability to evade RI. The trait ofevasion of RI is primarily responsible for the cytotoxicity of ONC andbovine seminal ribonuclease. It has also been found that RNasesuperfamily members, which are not natively cytotoxic, can be madecytotoxic by modifying their amino acid constituents, so as to inhibitbinding to RI.

Using the three dimensional structure of the porcine RI (pRI)-RNase Acomplex, RNase A was engineered to be more toxic to human leukemic cellsin vitro than ONC. Disruption of the RI•RNase A interface wasaccomplished by designing RNase A variants with amino acid substitutionsthat disrupted complementarity regions at the pRI•RNase A interface.These amino acid substitutions targeted short range pRI•RNase Ainteractions by incorporating sterically disruptive amino acids orremoving hydrogen bonds. This method is described in U.S. Pat. No.5,840,296, incorporated by reference herein in its entirety. Analogouscomplementarity regions were applied to bovine seminal ribonuclease(BS-RNase, 87% sequence similarity) a close homologue of RNase A.However, a BS-RNase variant with mutations at the same complementarityregions was less cytotoxic than ONC or the most cytotoxic RNase Avariant (D38R/R39D/N67R/G88R RNase A). This strategy did not result inthe level of cytotoxicity predicted for BS-RNase.

Furthermore, most of the work done so far in the creation of RNase Avariants has been done with bovine RNase A. However, the sequence andstructure of bovine RNase A (SEQ ID NO:1, GenBank Accession No.AAA72757) differs from human pancreatic ribonuclease 1 (RNase 1) (SEQ IDNO: 2, GenBank Accession No. CAG29314, incorporated by reference hereinin its entirety), RNase A and its homolog, RNase 1 share about 70%sequence identity of their amino acid sequences. While the bovineprotein may prove out to be acceptable for use in human therapy, aconservative approach might be to utilize a variant of a humanribonuclease, on the theory that use of a human protein might minimizecross-species antigenic problems. Accordingly, it is desirable to designvariants of human ribonucleases that may be more cytotoxic and effectivefor therapeutic, diagnostic or research use.

BRIEF SUMMARY OF THE INVENTION

The present invention is summarized as variants of human ribonuclease 1(RNase 1) identified through analysis of the interaction between RNase 1and the human ribonuclease inhibitor (hRI), as defined by the threedimensional (3-D) atomic structure of the hRI•RNase 1 complex.

In one aspect, the present invention defines an RNase 1 that hasimproved cytotoxic properties compared to all previously disclosedengineered ribonucleases.

In another aspect, the invention provides a variant RNase 1 having amodified amino acid sequence, wherein the variant RNase 1 retains itsribonucleolytic activity, and wherein the variant RNase 1 has a lowerbinding affinity for RI than that of the native RNase 1 and retainsnative ribonucleolytic activity.

In this aspect, the human RNase 1 variant includes at least two aminoacid changes from its native sequence, the changes causing evasion ofhuman hRI by RNase 1 through electrostatic repulsion, the first changebeing an amino acid substitution in the region of amino acid residues 85to 94 of RNase 1, and the second change being an alteration,substitution or amino acid swap at a location selected from the groupconsisting of amino acid residues 4, 7, 11, 31, 32, 38, 39, 41, 42, 66,67, 71, 111 and 118 of RNase 1, wherein the variant RNase 1 exhibitsenhanced cytotoxic activity relative to the native RNase 1.

In a related aspect, the human RNase 1 variant includes at least twoamino acid changes from its native sequence, the changes causing evasionof human hRI by RNase 1 through electrostatic repulsion, the firstchange being an amino acid substitution at amino acid residue 88 or 91of RNase 1, and the second change being an alteration, substitution oramino acid swap at a location selected from the group consisting ofamino acid residues 4, 7, 11, 31, 32, 38, 39, 41, 42, 66, 67, 71, 111and 118 of RNase 1, wherein the variant RNase 1 exhibits enhancedcytotoxic activity relative to the native RNase 1.

The present invention further provides variants of RNase 1 with aminoacids modified from the native sequence. Exemplary variants are providedin Table 5 herein below. Additional variants that have the desiredfunction are also within the scope of the invention.

In a preferred aspect, the RNase 1 variant is defined byR39D/N67D/N88A/G89D/R91D and has at least 10⁷-fold lower affinity and2700-fold lower association rate for hRI than wild-type (native) RNase1.

In another aspect, the present invention provides a method for modifyingthe amino acid sequence of a native RNase 1 to produce a novel,cytotoxic RNase 1.

The present invention is a method for modifying the amino acid sequenceof RNase 1 to produce a variant RNase 1, which retains itsribonucleolytic activity, and wherein the variant RNase 1 has a bindingaffinity for RI that is lower than that of the native RNase 1 andretains native ribonucleolytic activity.

The present invention is also a method for inhibiting the proliferationof cancer cells, comprising delivering to the cells an effective amountof a modified RNase 1, wherein the variant RNase 1 has a bindingaffinity for RI that is lower than that of the native RNase 1 andretains native ribonucleolytic activity.

In another aspect, the invention provides a method of engineeringcytotoxic RNase 1 variants by identifying electrostatic anchor residuesin the three dimensional structure of the hRI•RNase 1 complex; andmodifying the anchor residues identified in RNase 1 to inhibit bindingto hRI through electrostatic repulsion, wherein the variants retainnative ribonucleolytic activity, have a lower binding affinity for hRIthan that of the native RNase 1, and exhibit enhanced cytotoxic activityrelative to the native RNase 1.

In another aspect, the invention provides a crystal of a hRI•hRNase 1complex as defined by Protein Data Bank identification No. 1Z7X.

Also, disclosed is a method of using the three-dimensional structurecoordinates of the hRI*RNase 1 complex to design RNase 1 variants thatretain native ribonucleolytic activity, have a lower binding affinityfor hRI than that of the native RNase 1, and exhibit enhanced cytotoxicactivity relative to the native RNase 1.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although suitable methods andmaterials for the practice or testing of the present invention aredescribed below, other methods and materials similar or equivalent tothose described herein, which are well known in the art, can also beused.

Other objects, advantages and features of the present invention willbecome apparent from the following specification taken in conjunctionwith the accompanying drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A-B show RI contact residues of RNase 1 and RNase A. (A) Aminoacid sequence alignment of RNase A and RNase 1. (B) Three-dimensionalstructure of RNase 1 chain Z from PDB Identification No. 1Z7X.

FIGS. 2A-B show a color-coded comparison or the β4-β5 loop in RNase 1and RNase A when bound to RI.

FIGS. 3A-C show electron density at 1σ of key shape complementarityresidues between hRI and RNase 1.

FIGS. 4A-B show hRI-Affinity and cytotoxicity of RNase 1 and itsvariants. Legend for FIG. 4A is as follows: R39D/N67D/N88A/G89D (▪);N67D/N88A/G89D/R91D (Δ); R39D/N88A/G89D/R91D (●); R39D/N67D/N88A/R91D(◯); R39D/N67D/G89D/R91D (▾); R39D/N67D/N88A/G89D/R91D (□). Legend forFIG. 4B is as follows; D38R/R39D/N67R/G88R RNase A (Λ); G88R RNase A(◯); R39D/N67D/N88A/G89D/R91D RNase 1 (▴); R39L/N67L/N88A/G89L/R91LRNase 1 (●); N67D/N88A/G89D/R91D RNase 1 (♦); G38R/R39G/N67R/N88R RNase1 (▪); and wild-type RNase 1 (□).

FIGS. 5A-C show an electrostatic representation of the hRI and RNase 1interaction.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel human ribonuclease 1 variantsengineered to exhibit an increased level of cytotoxic activity relativeto the native RNase 1. This was made possible for the first lime throughthe determination of the three dimensional (3-D) atomic crystalstructure of the human ribonuclease inhibitor (hRI, SEQ ID NO: 4)molecule bound to the human ribonuclease 1 (RNase 1) molecule. Thestructure of the hRI•RNase 1 complex has a 1.95 Å resolution and theatomic coordinates were deposited in the publicly available sequencedatabase, Protein Data Bank (PDB), accession No. 1Z7X.

Using the 3-D structure of the hRI•RNase 1 complex, the interactionbetween hRI and RNase 1 in complex was characterized and used todetermine the energetic contribution of specific RNase 1 residues to RIbinding. The interaction between long range electrostatics and the rateof association was analyzed to identify electrostatic contributions ofanchor residues in the hRI•RNase 1 complex. These residues wererationally modified to (1) evade hRI by inhibiting the binding of theanchor residues through electrostatic repulsion and (2) increasecytotoxic activity relative to the native RNase 1. Using the logicdescribed here, it is believed that we were able to overcome a majorobstacle to the development of chemotherapeutics based on humanribonucleases.

In a broad embodiment, the invention provides an engineered ribonucleasevariant of RNase 1 having at least two amino acid changes from itsnative sequence, the changes causing evasion of hRI by RNase 1 throughelectrostatic repulsion, the first change being an amino acidsubstitution in the region of amino acid residues 85 to 94 of RNase 1,and the second change being an alteration, substitution or amino acidswap at a location selected from the group consisting of amino acidresidues 4, 7, 11, 31, 32, 38, 39, 41, 42, 66, 67, 71, 111 and 118 ofRNase 1, the variant RNase 1 having cytotoxic activity relative to thenative RNase 1. Such variants are designated herein by the notationXNNY, where Y is the substituted amino acid residue for the residue Xnormally found at location NN (e.g., R4C).

As used herein, the terms, “native”, “wild-type”, “unmodified” aresynonymous with each other. They refer to a gene product that has thecharacteristics of that gene product when isolated from a naturallyoccurring source. A wild-type gene is that which is most frequentlyobserved in a population and is thus arbitrarily designed the “normal”or “wild-type” form of the gene.

In contrast, the terms “variant”, “modified”, or “mutant” refer to agene product that displays modifications in sequence and or functionalproperties (i.e., altered characteristics) when compared to thewild-type gene product. The invention provides for variants of RNase 1Exemplary variants are described in Table 5.

In one embodiment, the invention provides a RNase 1 variant having anamino acid change at residues 4, 38, 39, 67, 88, 89, 91 an 118 causingevasion of human Ribonuclease Inhibitor (hRI) by RNase 1 throughelectrostatic repulsion relative to the native sequence, SEQ ID NO:2,wherein the variant RNase 1 retains its native ribonucleolytic activity,has a lower binding affinity for RI than that of the nativeribonuclease, and exhibits enhanced cytotoxic activity relative to thenative RNase 1.

Based on this embodiment, we synthesizedR4C/G38R/R39G/N67R/N88L/G89R/R91G/V118C RNase 1, which is an instructivevariant of human pancreatic ribonuclease. This enzyme has changes toeight residues, inspired by the atomic structure of the hRI•RNase 1complex as presented here. Residues Gly38, Arg39, Asn67, Asn88, Gly89,and Arg91 are all near the interface of the hRI•RNase 1 complex. Thereplacement of these residues in the variant is designed to interferewith the interface. Arg4 and Val118 are also near the hRI•RNase 1interface. Their replacement with cysteine residues is designed tointerfere with this interface as well as allowing for the formation of anew disulfide bond between Cys4 and Cys118, which is likely to conferextra conformational stability to the enzyme. This RNase 1 variant (1)retains nearly all of the enzymatic activity of the native RNase 1, (2)evades hRI, and (3) exhibits significant cytotoxic activity.

In a preferred embodiment, the invention provides RNase 1 variantshaving an amino acid change at residues 39, 67, 88, 89 and 91 relativeto the native sequence, SEQ ID NO:2, wherein the variants retain nativeribonucleolytic activity, have a lower binding affinity for RI than thatof the native RNase 1, and exhibit enhanced cytotoxic activity relativeto the native RNase 1.

These RNase 1 variants were designed by applying the relationshipbetween long range electrostatics and the rate of association. Apreferred RNase 1 variant is defined by R39D/N67D/N88A/G89D/R91D and has10⁷-fold lower affinity and 2700-fold lower association rate for hRIthan wild-type RNase 1. The 2700-fold lower association rate was 25-foldgain of electrostatic repulsion by aspartate substitution in RNase 1 and110-fold loss of electrostatic attraction at positions 39 and 91 inRNase 1.

It is noted that in the hRI•RNase 1 complex, electrostatic attraction ofkey charged residues like Arg39 and Arg91 helps hRI recognize RNase 1more than sterically constrained residues like Asn67 do. RI uses itshorseshoe shaped structure to allow long-range electrostaticinteractions by key solvent-exposed charged residues (especially Arg39and Arg91) to determine its tight interaction with RNase 1.

Other novel variants of RNase 1 are also described herein which retainnative ribonucleolytic activity, have a lower binding affinity for RIthan that of the native RNase 1, and exhibit enhanced cytotoxic activityrelative to the native RNase 1. These include RNase 1 variants, whichhave an amino acid change relative to the native sequence, SEQ ID NO:2at (1) residues 39, 67, 89 and 91, preferably, R39D/N67A/G89D/R91D; (2)residues 39, 67, 88 and 91, preferably R39D/N67D/N88A/R91D; (3) residues39, 67, 88 and 89, preferably R39D/N67D/N88A/G89D; (4) residues 39, 88,89 and 91, preferably R39D/N88A/G89D/R91D; (5) residues 38, 39, 67, and88, preferably G38R/R39G/N67R/N88R; and residues 67, 88, 89 and 91,preferably 67D/N88A/G89D/R91D.

In another embodiment, the invention provides method of modifying RNase1 to make cytotoxic RNase 1 variants by identifying electrostatic anchorresidues in the three dimensional structure of the hRI•RNase 1 complex;and modifying the anchor residues identified in RNase 1 to inhibitbinding to hRI through electrostatic repulsion, wherein the variantsretain native ribonucleolytic activity, have a lower binding affinityfor hRI than that of the native RNase 1, and exhibit enhanced cytotoxicactivity relative to the native RNase 1. Thus, through exploitation ofthe electrostatic attraction between hRI and RNase 1 we were able todevelop variants of RNase 1 that are capable of eluding the inhibitorybinding of RI to overcome a major hurdle in the development of humanribonuclease-based chemotherapeutics.

In another embodiment, the invention provides a method for inhibitingthe proliferation of cancer or tumor cells, comprising delivering to thecells an effective amount of a variant RNase 1, wherein the variantRNase 1 exhibits enhanced cytotoxic activity relative to the nativeRNase 1.

By “enhanced cytotoxicity” it is meant that the modified RNase 1exhibits greater cytotoxicity than the corresponding unmodified ornative (wild-type) RNase 1. In the examples below, cytotoxicity wasevaluated using the human erythroleukemia cell line K-562. It isanticipated that the modified ribonuclease of the present invention iscytotoxic against other tumor cells in addition to that which isdescribed herein. Inhibition of cell proliferation is determined bycalculating the percentage of viable K-562 cells treated with themodified or unmodified RNase 1, where 100% viability is considered to bethe number of viable cells that were treated with a solution ofphosphate-buffered saline (PBS).

By “effective amount” is meant that amount of ribonuclease needed tocause a significant reduction in the proliferation of the tumor cells.

Preferably, the modified ribonuclease reduces cell viability by at leastabout 10%. More preferably, the modified ribonuclease reduces cellviability by at least about 20%. Most preferably, the modifiedribonuclease reduces cell viability by about 50%, or even as much asabout 75%.

In another embodiment, the invention provides a hRI•RNase 1 complex asdefined by the Protein Data Bank identification No. 1Z7X. The 3-D atomiccrystal structure of the hRI•RNase 1 complex is described here for thefirst time. The atomic coordinates for the crystal structure of thehRI•RNase 1 complex are set forth here in Appendix 1. These atomiccoordinates were also deposited at the Research Collaborator) forStructural Bioinformatics Protein Data Bank (PDB) and assigned theaccession number PDB ID No: 1Z7X. The 3-D structure of the hRI•RNase 1complex can be used as a tool to rationally design RNase 1 variants thatevade hRI and that exhibit enhanced cytotoxic activity relative to thenative enzyme.

The research value of the 3-D structure of the hRI•RNase 1 complex isunderstood to those skilled in the art. It will also be appreciated thatthe structure obtained from X-ray crystallography is only a staticsnapshot of the protein-ligand complex. In reality, proteins like RNase1 are highly flexible macromolecules, changing their conformation onvarious time scales. Access to potential binding sites may only beavailable in certain conformations. It is envisioned that techniques,namely Molecular Dynamics, normal Mode or Monte Carlo methods, may beused to capture one or more representative structures for designingother RNase 1 variants.

While this patent specification contains several examples of protein andamino acid sequences, it should be understood that all protein sequencesare subject to minor changes and modifications without fundamentallychanging the proteins or the concept of the present invention.Conservative changes of amino acids of similar size and polarity arealways possible and rarely change the functioning of a protein. Thewhole RNase 1 is subject to further modifications of sequence, either byminor amino acid addition, deletion of substitution without adverselyaffecting the activity as a RNase 1. These kinds of changes in aminoacid sequence are interpreted to be within the scope of the languageused herein.

A conservative amino acid substitution includes one or more amino acidresidues within the sequence that can be substituted by another aminoacid of a similar polarity, which acts as a functional equivalent,resulting in a silent alteration. Substitutes for an amino acid withinthe sequence may be selected from other members of the class to whichthe amino acid belongs. For example, the nonpolar (hydrophobic) aminoacids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. Amino acids containingaromatic ring structures are phenylalanine, tryptophan, and tyrosine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine. The positively charged(basic) amino acids include arginine, lysine, and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Such alterations will not be expected to affect apparentmolecular weight as determined by polyacrylamide gel electrophoresis, orisoelectric point. Abbreviations of amino acids are known in the art

The invention is further clarified by consideration of the followingexamples, which are intended to be purely exemplary of the method of thepresent invention.

EXAMPLES 1. Experimental Overview Design of RNase 1 Variants

In general, it is understood by those skilled in the art that theequilibrium dissociation constant (K_(d)) of a protein complex isgoverned by the intermolecular factors influencing the rate ofassociation (k_(on)) and dissociation (k_(off)). The rate ofdissociation is influenced by factors that act over short distances,including van der Waals interactions, hydrogen bonds, hydrophobicinteractions, and salt bridges. The rate of association, however,depends primarily on diffusion but can be increased through Coulombicelectrostatic forces. The majority of long-range electrostatic forcesdestabilize protein-protein interactions due to a large energeticpenalty to desolvate the charged amino acids. However, when speed is anecessity, the rate of association and consequently the affinity of acomplex can be increased by optimizing the electrostatic energy.

In designing proteins to have lower affinity for their interactingprotein partner, either component of the kinetic rate (k_(on) ork_(off)) could be targeted. Previous inhibition studies of the RI•RNaseinterface have focused on short range intermolecular contacts betweenRI•RNase, effectively raising the dissociation rate. Detrimentallyaffecting the affinity of RNase 1, the human homologue of RNase A, usingshort range interactions has proven more difficult.

To overcome this hurdle, we determined the crystal structure of thehRI•RNase 1 complex at 1.95 Å resolution and employed the structuralinformation to design variants of RNase 1 with micromolar affinity forhRI (GenBank Accession No. P13489). We also investigated with RNase 1the analogous complementarity residues identified in RNase A andrevealed the energetic contribution to RI binding from these RNase 1residues. Based on the contribution of these charged residues (e.g.,Arg39 and Arg91) to the rate of complex association, we define a rolefor “electrostatic anchor” residues in determining protein-proteininteractions. Electrostatic anchor residues determine protein-proteinrecognition by (1) contributing substantial enhancement to theassociation rate, and (2) maintaining complex formation through tighthydrogen bonds. Overall, the evasion of RI by RNase 1 requires bothsteric and electrostatic contributions, but is driven to micromolaraffinity by a significant decrease in the association rate constant.

Accordingly, the cytotoxic RNase 1 variants of the present inventionwere developed by (1) analyzing the molecular recognition patterns of RIin complex with RNase 1 and RNase A, two ribonucleases with highsequence identity and (2) dissecting the difference in the energetic(e.g., steric and electrostatic) contribution of specific residuesinvolved in RI-binding to design rationally-based RNase 1 variants. Oneof the outcomes of this design strategy was engineering cytotoxic RNase1 variants with at least 10⁷-fold lower affinity and 2700-fold lowerassociation rate for hRI than wild-type RNase 1.

Differences in Pancreatic Ribonuclease Recognition by RI

The fast atomic density evaluator (FADE) algorithm revealed regions ofhigh shape complementarity between pRI and RNase A. By insertingdisruptive mutations in regions identified to have high shapecomplementarity, D38R/R39D/N67R/G88R RNase A (K_(d)=510 nM for hRI) andC31A/C32A/G38R/K39D/G88R BS-RNase (K_(d)=110 nM for hRI) were developedthat had significantly decreased affinities for RI. Using the same logicand shape complementarity regions, we designed G38R/R39G/N67R/N88R RNase1 (Table 5). When applied to RNase 1, this strategy failed to reduce theaffinity of hRI for RNase 1. The binding affinity of hRI for thisquadruple variant of RNase 1 was near the affinity of hRI for wild-typeRNase 1 Consequently, we wanted to determine what separated the RIrecognition of RNase 1 from RNase A.

Residue 39. Arg39 of RNase A had the highest shape complementarity scorefor pRI•RNase A and was proposed to be a secondary anchor residue. WhenArg39 was mutated to aspartate in G88R RNase A to create R39D/G88R RNaseA, the R39D mutation instilled 725-fold lower RI affinity In RNase 1,Arg39 has even lighter interactions with hRI with the formation of 3hydrogen bonds and consequently the energetic contribution of R39D issecond highest among the residues studied at ΔΔΔG=2.2 kcal/mol.

Similar to RNase A, a mutation of Arg39 to Asp decreases the affinity ofhRI for RNase 1, but the cytotoxicity of variants with R39D isproportionally lower than other variants with similar RI evasion. Partof the negative influence of an R39D substitution on the cytotoxicity ofRNase 1 variants can be accounted for by the 3-fold decrease in thecatalytic activity, but the increased activity does not completelyaccount for the high cytotoxicity of N67D/N88A/G89D/R91D RNase 1. Thedisproportionately large decrease in cytotoxicity in variants of RNase 1with R39D advocates a role for Arg39 in cell surface binding. Arg39 ispositioned between two positively charged patches on RNase 1 (FIG. 5)and so a negative charge at position 39 may weaken the cell surfacebinding of both positive patches, producing a proportionally greaterdecrease in internalization and cytotoxicity.

Residue 67. Previously, the recognition of Asn67 by hRI was exploited todevelop RI variants that selectively bind to angiogenin but not RNase 1or RNase A.₁₈ By incorporating a tryptophan at positions 408 and 410 inhRI, a highly selective variant of hRI was engineered that only boundangiogenin. A tryptophan substitution at Asn67 of RNase 1 to stericallyhinder the binding of residues 408-410 in hRI did not produce acomparable binding change (data not shown). Yet, an aspartate atposition 67 does destabilize the complex by 1.9 kcal/mol (Table 5).

Asn67 was proposed to be a primary anchor residue in the pRI-RNase Ainterface, due to its burial of surface area and its lack of molecularmotion.₂₂ In agreement with the assertion that Asn67 plays a role incomplex formation, we find that the energetic destabilization caused bymutation at position 67 is substantial (ΔΔΔG=1.9 kcal/mol). However,Arg39 and Arg91 provide more overall energy to stabilization of thehRI•RNase 1 complex.

β4-β5 loop. To determine what separated the RI recognition of RNase 1from RNase A in the β4-β5 loop region, a 3-D structural comparison wasperformed as shown in FIG. 2 between the β4-β5 loop in RNase 1 (purple)and RNase A (blue) when bound to RI (green). This was accomplished byaligning the alpha-carbons of RNase 1 and RNase A with the programSequoia, and images were created with the program PyMOL. (A) Structureof β4-β5 loops, with RI concealed. Side chains of residues 88-91 areshown as sticks. Amino acids are labeled with the color corresponding tothe color of the ribonuclease. (B) Orientation of the β4-β5 loop boundto RI. RNase A (chain E) was aligned to RNase 1 (chain Z) and thenmodeled into hRI (chain Y) based on the alignment to RNase 1. Hydrogenbonds are shown as dotted lines. Hydrogen bonds between hRI and RNase Aare hypothesized based on the alignment of hRI and pRI.

By performing this comparative alignment, we discovered that in contrastto prior results, where Gly 88 to arginine mutations decreased theaffinity of pRI for RNase A by 10⁴ M and the affinity of BS-RNase forhRI by 250-fold, substituting Asn88 with arginine in RNase 1 did notgenerate a similar decrease in affinity. In the crystal structure of thehRI•RNase 1 complex, the β4-β5 loop adopts a similar conformation toRNase A with pRI (FIG. 2). One major difference between RNase 1 andRNase A in the β4-β5 loop is with residue 88 where Asn88 of RNase 1hydrogen bonds with Glu264 instead of folding into the pocket formed byTrp261 and Trp263 like Gly88 in RNase A. Asn88 in RNase 1 is located onthe outer surface of the hRI•RNase 1 interface and could accommodate thestructural bulk of an arginine or carbohydrate chain while stillmaintaining high affinity for RI.

Gly89 of RNase 1 has been proposed to constitute the structural analogueof Gly 88 in RNase A, but mutational studies at Gly89 in RNase 1 have,also, failed to produce variants with lower affinity for RI. Gly89 inRNase 1 overlays more closely with Ser89 in RNase A (FIG. 2), but Gly89is unable to hydrogen bond with Glu206 as seen for Ser89 in RNase A.

Gly89 in RNase 1 still has van der Waals contact with Trp261 and Trp263in hRI•RNase 1, but Gly 89 appears to have greater flexibility thanGly88 in RNase A. Consequently, hRI can adjust to an aspartate orarginine substitution at Gly 89 in RNase 1 while maintaining nearwild-type affinity (FIG. 2).

Among the five residues investigated, Arg91 had the greatest energeticinfluence on the hRI•RNase 1 complex (ΔΔG=2.8 kcal/mol). Arg91 contactshRI in the negatively-charged bend of the hRI surface (FIG. 5), whereArg91 forms two hydrogen bonds with Glu287of hRI. Lys91 in RNase A wasproposed to play a secondary latching role in anchoring RNase A to pRI,but in RNase 1, Arg91 may serve as a primary anchor residue torecognition by hRI.

Substituting Arg91 with an aspartate severed the tight hydrogen bonds toGlu287of hRI, and replaced the attractive force of Arg91 with anelectrostatic repulsion. This loss of charge-charge attraction and gainof electrostatic repulsion at position 91 caused the largest change inthe overall binding affinity.

Electrostatic Anchor Residues

In a survey of 14 enzyme-inhibitor complexes, all fourteen complexes hada positive ΔG_(elec.) ³ meaning electrostatics were a negative force tocomplex formation. However, RI•RNase complexes are atypicalprotein-protein interfaces. Binding of angiogenin to hRI has anelectrostatic energy of interaction of −12.3 kcal/mol and a calculatedrate increase due to electrostatics of 10⁶M⁻¹s⁻¹. In contrast to othercomplexes, in RI•RNase complexes electrostatics play a key role inbinding.

FIG. 3 shows electron density at 1σ of key shape complementarityresidues between hRI (green) and RNase 1 (purple). Specific residuesshown in detail are (A) Arg39, (B) Asn67, and (C) Arg91. Highlightedregions are shown in wall-eyed stereo and interprotein hydrogen bondsare displayed by black dotted lines. Images were created with theprogram PyMOL, FIG. 3 demonstrates that instead ofsterically-constrained residues making initial contact with hRI, theelectrostatics of key solvent exposed charged residues like Arg39 andArg91 drive the association rate.

Specifically. Arg39 and Arg91 contributes at least 0.3 kcal/mol more tothe binding energy than Asn67, Asn88, or Gly89 do. The charged surfaceof Arg39 and Arg91 determines the association rate as substitution ofthese charges to leucine in R39L/N67L/N88A/G89L/R91L RNase 1 decreasesthe association rate by 110-fold. Hence, Arg39 and Arg91 serve a specialrole in the hRI•RNase 1 complex that we define as electrostatic anchorresidues. A residue that anchors the formation of a protein-proteincomplex should provide the major energetic force to complex formationand be the major marker for the recognition of its protein-bindingpartner.

Electrostatic residues like Arg39 and Arg91 fit these criteria, as theyare initially recognized by the electrostatic surface of hRI (FIG. 5).Specifically, FIG. 5 illustrates an electrostatic representation of thehRI (green) and RNase 1 (purple) interaction. Protein contact potentialof RNase 1, residues 39 and 91 are labeled (A), hRI (B), and hRI•RNase 1(C) as shown. The intensity of the blue (positive) and red (negative)coloration is indicative of the local electrostatic environment. Vacuumelectrostatics were calculated and images were created with the programPyMOL.

Furthermore, electrostatic residues Arg39 and Arg91 strongly affect theassociation rate of the complex (Table 6). Thus, Arg39 and Arg91 keepRNase 1 bound to hRI through tight hydrogen bonds (FIG. 3), allowingother contacts in the complex to form. Arg39 and Arg91 steer theformation of the hRI•RNase 1 complex over longer distances than thesterics of Asn67 and ultimately contribute more binding energy to theaffinity of the hRI•RNase 1 complex. Although, Arg39 and Lys91 in RNaseA were proposed to play a role in RI binding, the key function that theelectrostatics of these residues supply to the hRI•RNase 1 complex wasunderestimated.

Energetics of Evasion

Charged amino acids constitute 19% of all exposed amino acids on aprotein surface, but in the average protein-protein interface fewercharged residues are exposed. Charge-charge interactions inprotein-protein interfaces are disfavored energetically by a largeenergetic penalty to desolvate the exposed charge residue upon binding.The energetic penalty of desolvation can be circumvented by leaving keycharge interactions partially solvent exposed upon complex formation. InFIG. 3, the electron density for multiple solvent molecules are visiblesurrounding important charged interactions between hRI and RNase 1. RIseems to use its unusual horseshoe-shape to expose greater surface areato solvent and only partially desolvate key charged residues. Thisexposure to solvent diminishes the energetic desolvation penaltyincurred by RI upon RNase 1 binding and allows electrostatics to remaina driving energetic force to complex formation.

The positive charge on the RNase 1 surface (FIG. 5) facilitatessubstrate binding and consequently is necessary to maintain thebiological activity of RNase 1. RI takes advantage of the necessity fora charged surface on RNase 1 to tightly and rapidly inhibit RNase 1using long range electrostatics. FIG. 5 highlights the positive andnegative charge distribution on RNase 1 and hRI, respectively. In thecrystal structure, both Arg39 and Arg91 are tightly enclosed in thenegative inner surface of RI (FIG. 5) and anchor RNase 1 to the negativesurface of hRI.

By incorporating negatively-charged aspartate residues at keyelectrostatic anchors, we have lowered the equilibrium dissociationconstant of hRI for RNase 1 by nearly seven orders of magnitude.Comparison of variants of RNase 1 with mutations at Arg39 and Asn67illustrate how electrostatics help in the evasion of RI binding. Theelectrostatic repulsion of an aspartate at positions 39 and 67destabilizes the complex by 2.2 and 1.9 kcal/mol, respectively (Table5). If instead of an aspartate residue, the wild-type residue is changedto glycine at residue 39 and arginine at residue 67 (G38R/R39G/N67R/N88RRNase 1), a similar destabilization of the complex is not observed(total ΔΔG=3.0 kcal/mol). Thus, the electrostatics of residues Arg39 andAsn67 play a large role in determining the affinity of an RNase 1variant.

Overall, the repulsion of RI binding by aspartate substitutions in RNase1 is superadditive as the binding energy lost by the reversion of singlemutations in R39D/N67D/N88A/G89D/R91D RNase 1 (8.2 kcal/mol) is lessthan the binding energy lost with R39D/N67D/N88A/G89D/R91D RNase 1 (9.3kcal/mol). Examples of superadditive mutations in protein-proteincomplexes are uncommon, but have been observed with hRI•angiogenin. Thesuperadditive results for hRI•RNase 1, however, are surprising, becauseprevious mutations in hRI•RNase A were superadditive. Thesuperadditivity of the mutations to the hRI•RNase 1 complex can beexplained partially by the methods and partially by the type ofmutations. By combining multiple substitutions in RNase 1, the nativeRNase 1 structure may have been contorted such that additional mutationsdevelop disruptive contacts with RI that are not seen for singlemutants. Also, the electrostatic repulsion of an aspartate substitutioninstead of an alanine substitution can perturb a larger surface area andincrease the energetic destabilization of the mutation. For example, theΔΔΔG values for all four aspartate substitutions in RNase 1 are largerthan the ΔΔΔG value for deleting a single hydrogen bond (Asn88 to Ala88)(Table 5). Overall, we exploited the tight electrostatic attractionbetween hRI and RNase 1 to develop variants of RNase 1 with comparableaffinity to the most evasive RNase A variants.

Rates of Association and Dissociation

Electrostatics steer the formation of protein-protein complexes overlong distances and increase the rate of association over diffusionlimited processes. We measured the difference in the kinetic rateconstants between two variants of RNase 1 from Table 5 to determinewhich kinetic constant led to the increased evasion ofR39D/N67D/N88A/G89D/R91D RNase 1. Overall, changes in the dissociationrate (3100-fold) and association rate (2700-fold) constants ofR39D/N67D/N88A/G89D/R91D RNase 1 each account for half the decreasedaffinity for hRI as compared to wild-type RNase 1 (Table 6). Theimportant contribution of the association rate to the micromolaraffinity of R39D/N67D/N88A/G89D/R91D RNase 1 is seen more clearly whenits rates are compared to R39L/N67L/N88A/G89L/R91L RNase 1 (Table 6).

The 50-fold increased RI-evasion of R39D/N67D/N88A/G89D/R91D RNase 1over R39L/N67L/N88A/G89L/R91L RNase 1 is almost completely driven bylong-range electrostatic repulsion through its effect on the associationrate. The total influence of the electrostatics of residues 39, 67, 88,89, and 91 on hRI•RNase 1 complex formation can be approximated bycombining the 110-fold decrease in association rate due to the loss ofattractive forces by leucine substitutions and the 25-fold decrease inassociation rate with the gain of repulsive forces by aspartatesubstitutions. Overall, electrostatics contributes 2700-fold to thedecreased affinity of R39D/N67D/N88A/G89D/R91D RNase 1 for hRI and theexperimental results here reinforce previous calculations on theimportance of electrostatics in the binding of RI to ribonucleases. Theexample of hRI•RNase 1 demonstrates that proteins have evolved anadditional strategy, using electrostatic anchor residues, forrecognizing their protein partner in solution when charge stronglyinfluences the association rate.

These results demonstrate that cytotoxic variants of human RNase 1 arequite possible to construct based on the data revealed here. Weanticipate that consideration of the atomic coordinates of the hRI•RNase1 complex can lead to even more cytotoxic variants of RNase 1.

2. Detailed Methods and Materials

Materials: Escherichia coli BL21 (DE3) and pET22b(+) were from Novagen(Madison, Wis.). The fluorogenic ribonuclease substrate,6-FAM-dArU(dA)2-6-TAMRA, was from Integrated DNA Technologies(Coralville, Iowa). Enzymes were from Promega (Madison, Wis.). K-562cells were from the American Type Culture Collection (Manassas, Va.).Cell culture medium and supplements were from Invitrogen (Carlsbad,Calif.). [methyl-3H]Thymidine (6.7 Ci/mmol) was from Perkin-Elmer(Boston, Mass.). HiTrap NHS-ester columns were from Amersham Biosciences(Piscataway, N.J.). RNase A Type III-A for attachment to HiTrapNHS-ester columns was from Sigma-Aldrich (St. Louis, Mo.). MES buffer(Sigma-Aldrich, St. Louis, Mo.) was purified by anion exchangechromatography to remove trace amounts of oligomeric vinylsulfonic acid.All other chemicals were of commercial grade or better, and were usedwithout further purification. Terrific Broth (TB) contained (in 1.00 L)tryptone (12 g), yeast extract (24 g), glycerol (4 mL), KH2PO4 (2.31 g),and KH2PO4 (12.54 g). Phosphate-buffered saline (PBS) pH 7.4 contained(in 1.00 L) NaCl (8.0 g), KCl (2.0 g). Na2HPO4.7H20 (1.15 g), KH2PO4(2.0 g), and NaN3 (0.10 g).

Instrumentation: Fluorescence measurements were made with aQuantaMaster1 photoncounting fluorimeter with sample stirring (PhotonTechnology International, South Brunswick, N.J.). Thermal denaturationdata were collected using a Cary 3 double-beam spectrophotometerequipped with a Cary temperature-controller (Varian, Palo Alto, Calif.).[methyl-3H]Thymidine incorporation into genomic DNA was quantified byliquid scintillation counting using a Microbeta TriLux liquidscintillation and luminescence counter (Perkin-Elmer, Wellesley, Mass.).The mass of RNase 1 and its variants was confirmed by matrix-assistedlaser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometryusing a Voyager-DEPRO Biospectrometry Workstation (Applied Biosystems,Foster City, Calif.).

RNase 1 purification: RNase 1 was purified from inclusion bodies usingthe same oxidative refolding procedure described previously.₁₉ Variantsof RNase 1 were created by Quikchange site-directed mutagenesis orQuikchange Multi site-directed mutagenesis (Stratagene, La Jolla. CA)following the manufacturer's protocol. Variants were purified using thesame procedure used for wild-type RNase 1.₁₉ Variants of RNase 1 withfree cysteine residues at position 19 were protected with5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) before fluorophoreattachment. Then, immediately before use, TNB-protected variants weredeprotected using a three-fold molar excess of dithiothreitol (DTT) anddesalted by chromatography using a PD-10 desalting column (AmershamBiosciences, Piscataway, N.J.). RNase 1 conjugates with 5-iodoacetamidofluorescein (Sigma-Aldrich, St. Louis, Mo.) were prepared by reactionwith a ten-fold molar excess of 5-iodoacetamido fluorescein for 4-6 h at25° C. Conjugates were purified by chromatography using a HiTrap SP FFcolumn. The molecular masses of RNase 1 and its variants were confirmedby matrix-assisted laser desorption/ionization time-of-flight(MALDI-TOF) mass spectrometry using a Voyager-DEPRO BiospectrometryWorkstation.

hRI purification: hRI was purified similarly to procedures describedpreviously. Briefly, a pET-22b(+) plasmid that contained cDNA for hRIwas transformed into E. coli BL21(DE3) and a single colony was used toinoculate LB medium (25 mL) containing ampicillin (150 μg/mL). A starterculture was grown for 16 h at 37° C. and 250 rpm and was used toinoculate cultures of TB medium (1.00 L) containing ampicillin (200μg/mL).

These cultures were grown at 37° C. and 225 rpm until OD_(o00)>3.0.Expression of the hRI cDNA was induced by adding IPTG (0.5 mM) andgrowing for 16 h at 18° C. and 225 rpm. Bacteria were collected bycentrifugation (12,000×g for 10 min) and resuspended in 30 mL of 50 mMTris-HCl buffer, pH 7.5, containing EDTA (10 mM) and DTT (10 mM).Bacteria were lysed by two passes through a French pressure cell, andthe cellular debris was removed by ultracentrifugation. RNase A wasattached covalently to the resin in two 5-mL HiTrap NHS-ester columns,following the manufacturer's protocol. The supernatant was loaded ontothese two columns connected in series. The peak eluted from the RNase Aaffinity columns was dialyzed for 16 h against 4 L of 20 mM Tris-HClbuffer, pH 7.5, containing DTT (10 mM) and EDTA (1 mM) and purifiedfurther by chromatography using a HiTrap Q column.₄₆ The purity of theeluted hRI was shown to be > 99% by SDS-PAGE (data not shown).

Complex purification: Purified RNase 1 (50 mg/mL) and hRI (10 mg/mL)were mixed at a molar ratio of 1.2 to 1.0, respectively. This solutionwas incubated at 25° C. for 60 min to allow for complex formation. Thecomplex was loaded onto a 5-mL HiTrap Q column that had beenpre-equilibrated with 20 mM Hepes-NaOH buffer, pH 7.5, containing DTT(10 mM) and glycerol (2% v/v). The complex was eluted with a lineargradient of NaCl (0-0.4 M) over 30 column volumes. Free RNase 1 elutedwith the flowthrough, and the hRI•RNase 1 complex eluted at about 0.15 MNaCl. Purified complex was dialyzed for 16 h at 4° C. against 20 mMHepes-NaOH buffer, pH 7.5, containing DTT (10 mM) and glycerol (2% v/v).Finally, the complex was concentrated in a Vivaspin 20 mL centrifugalconcentrator (Vivascience AG, Hannover, Germany) at 6,000×g to a finalconcentration of 10 mg/mL. Aliquots were flash frozen and stored at −80°C.

Crystallization: Crystals of the hRI•RNase 1 complex were obtained byhanging-drop vapor diffusion in 20 mM sodium citrate buffer, pH 4.2,containing methyl ether PEG 2000 (10% w/v), ammonium sulfate (1 mM), andDTT (25 mM) with the hanging drop solution containing a mixture ofpurified hRI RNase 1 (0.9 μL) and citrate buffer solution (5.1 μL).Diffraction-quality crystals grew within a week at 25° C. Proteincrystals were soaked in reservoir solutions containing increasingamounts of ethylene glycol up to 25% (v/v), and were flash-cooled in astream of cryogenic N₂(g).

Diffraction data were collected at SER-CAT Sector 22 at Argonne NationalLaboratories. The crystal was maintained at 100 K during datacollection, and X-rays were tuned to a wavelength of 0.99997 Å. Thediffraction images were integrated and scaled using HKL2000. The phaseswere determined through molecular replacement using MOLREP from the CCP4suite with PDB entry 1DFJ as the starting model. Arp-Warp₅₀ was used tobuild the initial model, which was then completed with alternate cyclesof model building with Xfit₅₁ and refinement using REFMAC. Thestructural coordinates for the x-ray structure of human ribonucleaseinhibitor complexed with ribonuclease inhibitor have been deposited inthe Protein Data Bank (PDB) having an accession or identification No.1Z7X, incorporated by reference herein in its entirety.

Ribonucleolytic activity: The ribonucleolytic activity of RNase 1 andits variants was quantitated using 6-FAM-dArU(dA)₂-6-TAMRA. Cleavage ofthis substrate at the uridine ribonucleotide leads to a 180-foldincrease in fluorescence Assays were carried out at 23(±2)° C. in 2 mlof 0.10 M Mes-NaOH buffer, pH 6.0, containing NaCl (0.10 M).Fluorescence data were fitted to the equation:k_(cat)/K_(M)=(ΔI/Δt)/((I_(f)−I₀)[E]) where ΔI/Δt represents the initialreaction velocity, I₀ is the fluorescence intensity before the additionof a ribonuclease, I_(f) corresponds to final fluorescence aftercomplete substrate hydrolysis, and [E] is the total ribonucleaseconcentration.

Conformational stability: The conformational stability was determined byfollowing the change in absorbance at 287 nM with increasingtemperature. The temperature of PBS containing a ribonuclease (0.1-0.2mg/mL) in PBS was raised from 20 to 80° C. at 0.15° C./min. The A₂₈₇ wasfollowed at 1° C. intervals and the absorbance change were fitted to atwo-state model of denaturation, in which the temperature at themidpoint of the transition curve corresponds to T_(m).

RI evasion: The affinity of RNase 1 variants for hRI was determined byusing a fluorescent competition assay reported previously with minormodifications. Briefly 2.0 mL of PBS containing DTT (5 mM),fluorescein-labeled G88R RNase A (50 nM), and an unlabeled RNase 1variant was incubated at 23 (±2)° C. for 20 min. The initialfluorescence intensity of the unbound fluorescein-labeled G88R RNase Awas monitored for 3 min (excitation: 491 nm; emission: 511 nm) hRI wasthen added to 50 nM and the final fluorescence intensity was measured.Values for K_(d) were obtained by nonlinear least-squares analysis ofthe binding isotherm using the program DELTAGRAPH 5.5 (Red RockSoftware, Salt Lake City, Utah). The K_(d) value for the complex of hRIand fluorescein-labeled G88R RNase A is 1.4 nM.

Kinetic assay: The dissociation rate constant for complexes of hRI andvariants of RNase 1 were determined by a procedure similar to thatdescribed previously. Briefly, equimolar concentrations of hRI andfluorescein-labeled RNase 1 variant were allowed to reach equilibrium inPBS containing DTT (5 mM). The equimolar concentrations were 20-foldgreater than the previously determined K_(d) value for each hRI•RNase 1complex. After reaching equilibrium, a 100-fold molar excess ofwild-type RNase A (Sigma-Aldrich) was added to scavenge free hRI. Theincrease in fluorescence was followed as the hRI•RNase 1 variant complexdissociated irreversibly. To calculate the dissociation rate constant,k_(d), the data were fitted to eq 1, wherein F₀ is the fluorescencebefore the addition of wild-type RNase A and F₀₀ is the fluorescenceafter complete dissociation of the complex.F=F ₀+(F ₀₀ −F ₀)(1−e ^(kdt))  (1)

Cytotoxicity: The effect of RNase 1 and its variants on theproliferation of K-562 cells was assayed as described previously.Briefly, after a 44-h incubation with ribonuclease, K-562 cells weretreated with [methyl-³H]thymidine for 4-h and the incorporation ofradioactive thymidine into the cellular DNA was quantified by liquidscintillation counting. Results are shown as the percentage of[methyl-³H]thymidine incorporated into the DNA as compared to theincorporation into control K-562 cells where only PBS was added. Dataare the average of three measurements for each concentration, and theentire experiment was repeated in triplicate. Values for IC₅₀ werecalculated by fitting the curves using nonlinear regression to eq 2,wherein y is the total DNA synthesis following the [methyl-³H]thymidinepulse, and h is the slope of the curve.

$\begin{matrix}{y = \frac{100\%}{1 + 10^{{({{\log{({IC}_{50})}} - {\log{\lbrack{ribonuclease}\rbrack}}})}h}}} & (2)\end{matrix}$

RESULTS

Important Interactions Between hRI and RNase 1

RNase 1 and RNase A share 70% sequence identity, but previousmutagenesis studies have suggested a variation in how they arerecognized by RI. To structurally elucidate these differences in RIbinding, crystals of the hRI•RNase 1 complex were grown under low ionicconditions as described herein below. The structure was refined to anR-value of 0.175 (R-free 0.236) and at a resolution of 1.95 Å (Table 1).

TABLE 1 Crystallographic, data processing, and refinement statistics.Data Collection Statistics Native Space Group P212121 Unit CellParameters a = 71.338, b = 107.546, c = 155.036 alpha beta gamma 90.0090.00 90.00 Energy (keV) 12.399 Wavelength (Å) 0.99997 OverallResolution Range (Å) 47.17-1.95 (2.00-1.95)  Number of ReflectionsMeasured 573939, Unique 84446 Completeness (%) 97.0 (72.6) Rmerge^(a)0.078 (0.424) Redundancy 6.8 (3.6) Mean I/σ (I) 16.96 (2.94)  Phasing MRCorrelation Coefficient (MOLREP) 0.223 MR Model IDFJ Refinement andModel Statistics from REFMAC 5.2.0005 Data Set Native Number ofreflections (Total) 80141 Number of reflections (Free) 4225 R_(cryst)^(b) (R_(free) ^(c)) 0.175 (0.236) RMSD bonds (Å) 0.016 RMSD angles (°)1.515 ESU based on R_(free) (Å) 0.166 Average B factor (Å²) 28.04 Numberof water molecules 854 Ramachandran plot Residues in most favorableregion 86.8% Residues in additional allowed region 12.8% Residues ingenerously allowed region 0.4% Residues in disallowed region 0.0% Valuesin parentheses refer to the highest resolution shell. ^(a)R_(merge) =3_(h)3_(i) *I_(i)(_(h)) − <I(h)>/3_(h)3_(i)/_(i)(h), where I_(i)(h) isthe intensity of an individual measurement of the reflection and <I(h)>is the mean intensity of the reflection. ^(b)R_(cryst) = 3_(h)**F_(obs)* − *F_(calc) * */3_(h)*/F_(obs)*, where F_(obs) and F_(calc)are the observed and calculated structure-factor amplitudes,respectively. ^(c)R_(free) was calculated as R_(cryst) using 5.0% of therandomly selected unique reflections that were omitted from structurerefinement.

Tables 2, 3, and 4 summarize some of the results of the analysis of theraw data, which was included in Appendix A of the corresponding U.S.priority application Ser. No. 60/691,311. The atomic coordinates werealso submitted to the protein Data Bank (Accession No. 1Z7X). Table 3lists data from the analysis of the interaction between hRI and RNase 1,and identifies those amino acid residues in the human RNase 1 structurewhich are less than 3.20 Angstroms from amino acid residues in hRI whenRNase 1 is bound to hRI. The distance of 3.20 Angstroms is a maximaldistance for the existence of a meaningful interaction between the twomolecules and thus indicates residues in RNase 1 that can be substitutedto alter the interaction between the two molecules. This list includesseveral of the residues, the variations in which have demonstratedconversion of RNase A into a cytotoxic molecule, notably residue 88.

TABLE 2 RNase 1 Residues ≦3.20 Å from hRI Residues in hRI•RNase 1Complex Arg4 Pro42 Lys7 Lys66 Gln11 Asn71 Arg31 Asn88 Arg32 Arg91 Arg39Glu111 Lys41

Tables 3 and 4 list the locations of closest interaction between hRI andhuman RNase 1, as revealed by analysis of the atomic locations of themolecules in the two distinct molecular complexes (W—X and Y—Z) thatformed the crystal used in the structural analysis below. The 3-Dstructures of these two molecular complexes were similar, but notidentical, as can be seen from Table 3 and Table 4.

TABLE 3 W•X Complex RNase 1 (X) atom hRI (W) atom distance (Å) Arg4 NH1Ala441 CB 2.33 Lvs7 CE Ser461 OXT 3.18 Gln11 NE2 Ser461 OXT 3.05 Arg31NH1 Gln11 OE1 2.78 Arg31 NH2 Arg34 NH1 2.89 Arg32 NE Asp37 OD2 2.78Arg39 NE Glu402 OE2 2.39 Arg39 NE Trp376 CH2 3.11 Lys41 CE Asp436 OD12.83 Lys66 NZ Asn407 OD1 2.87 Asn71 ND2 Tyr438 OH 2.61 Asn88 OD1 Glu265OE2 2.72 Arg91 NH2 Glu288 OE2 2.65

TABLE 4 Y•Z Complex RNase 1 (Z) atom hRI (Y) atom distance (Å) Lys7 NZGlu444 OE2 3.18 Arg32 NE Asp37 OD1 2.63 Arg39 NE Glu402 OE2 2.79 Lys41NZ Asp436 OD1 2.68 Pro42 CG Asn407 ND2 3.17 Lys66 CE Cys409 SG 3.20Asn71 ND2 Tyr438 OH 2.86 Asn88 OD1 Glu265 OE2 2.70 Arg91 OD1 Glu288 OE22.60 Glu111 OE2 Tyr438 OH 2.58

From this summary of the raw data, it can be understood that amino acidresidues Arg39, Asn88, and Arg91 represent prime locations for modifyingRNase 1 to interfere with the binding of hRI. As a result, RNase 1 wouldbe able to evade the action of the inhibitor in vivo and increasecytotoxicity of RNase 1 for chemotherapeutic purposes.

The contacts from both chains of RNase 1 in the unit cell are providedin FIG. 1A The secondary structure of RNase A is identified with h(α-helix), s (β-strand), or t (turn). Residues in van der Waals orhydrophobic contact with RI are in blue. Residues with hydrogen bonds toRI are in red. Conserved cysteine residues are in yellow. Key catalyticresidues are in black boxes. In chain X of RNase 1 (FIG. 1 a), a boundcitrate molecule forms tight hydrogen bonds to all three catalyticresidues, His 12, His 118 and Lys41. The bound citrate perturbs thesubstrate binding cleft of RNase 1 causing Arg10 and Lys66 to undergosignificant conformational changes. Lys66 in chain X forms a hydrogenbond to Asn406 of hRI. However, in chain Z (FIG. 1B, 3-D structure ofRNase 1 chain Z from 1Z7X), only van der Waals interactions wereobserved with hRI. Residues are colored using the same scheme as in thesequence alignment in FIG. 1A, except that active-site residues are nothighlighted. The image was created with the program PyMOL (DeLanoScientific, South San Francisco, Calif.). To facilitate comparisonbetween the structures of hRI•RNase 1 and pRI•RNase A,₁₅ chains Y (hRI)and Z (RNase 1) (without citrate bound) serve as the hRI•RNase 1 complexfor comparison.

The root mean square deviation (rmsd) between the alpha carbons ofhRI•RNase 1 and pRI•RNase A is 2.8 Å. Much of the deviation between thecomplexes originates from the alignment of pRI with hRI (rmsd=1.6 Å),because unlike hRI, pRI is observed to undergo a conformational changeupon RNase A binding. The alpha carbons of RNase 1 and RNase A have lessdeviation (rmsd=0.6 Å). Angiogenin and EDN, the other humanribonucleases crystallized with hRI, gave rmsd values of 7.4 and 6.3 Åfrom RNase 1, respectively. The considerably higher rmsd for angiogeninand EDN shows the structural variation among human ribonucleases andunderscores the similarity between RNase 1 and RNase A.

The conservation of contact residues between the complexes of hRI•RNase1 and pRI•RNase A is shown in FIG. 1A. The localization of RI-contactresidues on the active-site face of RNase 1 is shown in FIG. 1B. Thetotal number of contact residues (23) with RI is conserved between RNase1 and RNase. A divergence in the recognition of RNase 1 and RNase A byRI is in the number of ribonuclease residues observed to form a hydrogenbond to RI. In RNase 1, 13 residues form at least one hydrogen bond toRI, compared to 8 in RNase A.

Previous studies on RNase A and BS-RNase binding to RI focused on threestructural regions, residues 38/39, residue 67, and residues in the(β4-β5 loop. FIGS. 2 and 3 emphasize the hydrogen bonding network andelectron density of these regions for the hRI•RNase 1 complex. Arg39 ofRNase 1 forms three hydrogen bonds with hRI that are absent with Arg39in RNase A. Arg39 of RNase 1 makes a bidentate hydrogen bond with Glu401and a main-chain hydrogen bond to Tyr434 of hRI. The hydrogen bondformed from Asn67 to RI shifts from Val405 (Leu409 in hRI) in pRI•RNaseA to Tyr437 in hRI RNase 1. The β4-β5 loop forms hydrogen bondsinvolving Asn88, Gly89, and Arg91 (FIG. 2). Arg91 of RNase 1 forms twotight hydrogen bonds (< 3.0 Å) from the nitrogens in its guanidino groupto Glu287 in hRI (FIG. 3).

In contrast, Lys91 in RNase A is directed away from Glu287 in pRI and nohydrogen bonds were observed (FIG. 2).₁₅ Based on the structuralenvironment of the complementarity regions, two variants of RNase 1 weredesigned. One variant of RNase 1 (G38R/R39G/N67R/N88R RNase 1) mimicsthe most cytotoxic variant of RNase A (D38R/R39D/N67R/G88R RNase A), byswapping the amino acids in positions 38/39 and by incorporating bulkyresidues at positions 67 and 88. The other RNase 1 variant(R39D/N67D/N88A/G89D/R91D RNase 1) utilizes the same regions, butinstead of steric bulk utilizes electrostatic repulsion to inhibit thebinding of RI.

Ribonucleolytic Activity

The ability of a ribonuclease to cleave RNA in the presence of RI isclosely correlated to its cytotoxicity in vitro. For a ribonucleasevariant to achieve its full cytotoxic potential, a mutation thatdecreases RI binding must not detrimentally affect the native catalyticactivity.₂₅ Consequently, variants of RNase 1 were assayed for theircatalytic activity toward a tetranucleotide substrate. Values ofk_(cat)/K_(M) for RNase A, RNase 1, and their variants are given inTable 5. The k_(cat)/K_(M) value for wild-type RNase 1 is 10-fold higherthan the value previously reported. A similar increase in the catalyticactivity was measured for RNase A when oligovinyl sulfonate, a potentinhibitor of ribonuclease, was removed from the reaction buffer.

TABLE 5 Biochemical parameters of RNase 1, RNase A, and their variants.k_(cat)/K_(M) ^(b) ΔΔG^(d) ΔΔΔG^(e) Ribonuclease Tin^(a) (° C.) (10⁶M⁻¹s⁻¹) K_(d) ^(c) (nM) (kcal/mol) (kcal/mol) IC₅₀ ^(f) (μM) Z Wild-typeRNase A  64^(g) 52 ± 4^(g)   44 × 10^(−6h) >25 +4 D38R/R39D/N67R/ 56^(g) 38 ± 6^(g)   510 ± 30^(g) 0.15 ± 0.01 +6 G88R RNase A Wild-typeRNase 1 57 21 ± 2    20 × 10^(−5i) >25 +6 G38R/R39G/N67R/ 61 4.2 ± 0.40.032 ± 0.016 3.0 >25 +8 N88R RNase 1 R39D/N67D/N88A/ 58 6.3 ± 0.5 (1.7± 0.5) × 10³ 9.5 13.3 ± 1.7  0 G89D/R91D RNase 1 R39L/N67L/N88A/ 65 30 ±3    30 ± 1 7.1 2.4 >25 +4 G89L/R91L RNase 1 N67D/N88A/G89D/ 51 16 ± 6   45 ± 15 7.3 2.2 >25 +2 R91D RNase 1 R39D/N88A/G89D/ 57 10 ± 3    68 ±8 7.6 1.9 >25 +1 R91D RNase 1 R39D/N67D/G89D/ 54 3.3 ± 0.5 (1.0 ± 0.1) ×10³ 9.1 0.4 >25 0 R91D RNase 1 R39D/N67D/N88A/ 51 10 ± 1    278 ± 50 8.41.1 >25 +1 R91D RNase 1 R39D/N67D/N88A/ 57 5 ± 1   16 ± 3 6.7 2.8 >25 +2G89D RNase 1 ^(a)Values of k_(cat)/K_(M) (±SE) were determined forcatalysis of 6-FAM-dArU(dA)₂-6-TAMRA cleavage at 25° C. in 0.10 MMES-NaOH buffer (OVS-free), pH 6.0, containing 0.10 M NaCl (9).^(b)Values of T_(m) (±2° C.) for RNase 1 and its variants weredetermined in PBS by UV spectroscopy. ^(c)Values of K_(d) (±SE) weredetermined for the complex with hR1 at 25° C. (10) ^(d)Values of ΔΔGwere calculated with the equation: ΔΔG = −RTln(K_(d) ^(wild-type)/K_(d)^(variant)). ^(e)Values of ΔΔΔG = ΔΔG^(R39D/N67D/N88A/G89D/R91D RNase 1)− ΔΔG^(RNase 1 variant). ^(f)Values for IC₅₀ (±SE) are for incorporationof [methyl-³H]thymidine into the DNA of K-562 cells treated with theribonuclease. and were calculated with eq 1. ^(g)From Rutkoski et al.^(h)From Lee et al. ^(i)From Saxena et al.

The k_(cat)/K_(M) values for all variants of RNase 1 are within 6-foldof the wild-type enzyme. Unlike RNase A, substitutions at residues38/39, residue 67, and the residues in the β4-β5 loop of RNase 1 candetrimentally affect the catalytic activity. The influence of theseresidues is observed in the 5-fold and 3.3-fold decrease in activity ofG38R/R39G/N67R/N88R RNase 1 and R39D/N67D/N88A/G89D/R91D RNase 1,respectively. An anomaly to this trend is R39L/N67L/N88A/G89L/R91L whosecatalytic activity is unaffected by substitutions at these residues. Thediscrepancy could result from a compensating favorable hydrophobicinteraction between the substituted leucines and the substratenucleotide bases, although none of the positions mutated were previouslyproposed to be involved in substrate binding.

By reverting only one substitution in R39D/N67D/N88A/G89D/R91D RNase 1to the wild-type amino acid (Table 5), the contribution of individualmutations to the k_(cat)/K_(M) value can be deduced. For example inR39D/N67D/N88A/G89D/R91D RNase 1, an aspartate residue at position 39decreases the activity 2.5-fold with respect to N67D/N88A/G89D/R91DRNase 1. Substitutions of N67D or G89D are responsible for a 1.6-folddecrease in the k_(cat)/K_(M) value, where as mutations of R91D and N88Alead to a 1.3-fold and 1.9-fold increase in the k_(cat)/K_(M) value,respectively. The contribution to the catalytic activity of eachsubstitution in R39D/N67D/N88A/G89D/R91D RNase 1 seems to be additive asthe total change in the k_(cat)/K_(M) value for all five singlesubstitutions (2.6-fold) approaches the 3.3-fold reduction in thek_(cat)/K_(M) value for R39D/N67D/N88A/G89D/R91D RNase 1.

In regard to R4C/G38R/R39G/N67R/N88L/G89R/R91G/V118C RNase 1, it wasfound to retain nearly all of the enzymatic activity of the wild-typeenzyme, having a k_(cat)/K_(M) value of (1.4±0.8)×10⁶ M⁻¹s⁻¹ undersimilar assay conditions.

Thermal Stability

The thermal stability of a ribonuclease is linked to its susceptibilityto proteolysis and consequently its cytotoxicity. The T_(m) values forall RNase 1 variants are shown in Table 5. The T_(m) value of wild-typeof RNase 1 is close to the previously reported value. In agreement withprevious studies, incorporation of charged patches on the surface ofRNase 1 does not reduce the T_(m) value by more than 6° C. Neitherarginine nor aspartate substitutions at residues 38/39, residue 67, orresidues in the β4-β55 loop significantly disturb the conformationalstability, as G38R/R39G/N67R/N88R RNase 1 and R39D/N67D/N88A/G89D/R91DRNase 1 have T_(m) values comparable to wild-type RNase 1 (61 and 58°C., respectively.) The largest change in the conformational stability isobserved with certain combinations of aspartate substitutions. Forinstance, N67D/N88A/G89D/R91D and R39D/N67D/N88A/R91D decrease the T_(m)value by 6° C. and R39D/N67D/G89D/R91D by 3° C. Each of these RNase 1variants has substitutions of both N67D and R91D, where as variants withonly an N67D or R91D substitution have wild-type stability. Positions 67and 91 are located on opposite sides of the RNase 1 active site, so anexplanation for their synergistic contribution to thermal stability willrequire further study. Overall, all variants of RNase 1 are stable wellabove physiological temperature.

Evasion of Ribonuclease Inhibitor

RI binds multiple members of the RNase A superfamily with equilibriumdissociation constant values in the femtomolar range, forming one of thetightest noncovalent biological interactions. By mutating residues38/39, 67, and 88 in RNase A (D38R/R39D/N67R/G88R RNase A), theequilibrium dissociation constant of the hRI•RNase A complex wasincreased by seven orders of magnitude (Table 5). The analogous variantin RNase 1 (G38R/R39G/N67R/N88R RNase 1) maintained near wild-typeaffinity (Table 5). However, substituting the arginine residues inG38R/R39G/N67R/N88R RNase 1 with multiple aspartate residues and onealanine residue reduces the affinity of RI for RNase 1 by nearly10⁷-fold. The K_(d) value for R39D/N67D/N88A/G89D/R91D RNase 1 (1.7 μM)is close to the highest measured for any RNase A variant (2.9 μM). Whenthe aspartate substitutions in R39D/N67D/N88A/G89D/R91D RNase 1 arereplaced with the isosteric amino acid, leucine(R39L/N67L/N88A/G89L/R91L RNase 1), the equilibrium dissociationconstant increases 50-fold, Leucine substitution causes the disruptionof 7 kcal/mol of RI-binding energy by the loss of electrostaticattraction and steric hindrance, but an additional 2.4 kcal/mol ofbinding energy is disturbed by the electrostatic repulsion of anaspartate residue at the same positions.

The influence of electrostatics on RI evasion is further expanded inTable 6, where the individual kinetic rate constants for the complex ofhRI and two fluorescein-labeled RNase 1 variants are shown. Thedissocation rate increases 1400-fold over wild-type RNase 1 uponsubstitutions of R39L/N67L/N88A/G89L/R91L in RNase 1, but remains nearlyconstant (2-fold increase) upon aspartate substitution(R39D/N67D/N88A/G89D/R91D RNase 1). The association rate is affectedmore proportionally by both leucine substitution (110-fold decrease) andby aspartate substitutions (25-fold decrease). The substantial change inthe association rate with both leucine and aspartate demonstrates thetwo electrostatic forces that can lower the km value, loss of attractiveforces (leucine substitution) and gain of repulsive forces (aspartatesubstitution). Overall, the nearly 10⁷-fold decrease in binding affinityof hRI for R39D/N67D/N88A/G89D/R91D RNase 1 contains equal contributionsby the dissociation rate (3100-fold) and association rate (2700-fold).Yet, the additional 50-fold decrease in the K_(d) value ofR39D/N67D/N88A/G89D/R91D RNase 1 over R39L/N67L/N88A/G89L/R91L RNase 1is driven by a decreased association rate.

TABLE 6 Contribution of the kinetic rate constants to overall R1 bindingaffinity Ribonuclease k_(off) (s⁻¹) ^(a) k_(on) (M⁻¹s ⁻¹ ) ^(b)wild-type 6.8 × 10^(−5c) 3.4 × 10^(8d) RNase 1 R39D/N67D/N88A/  0.22 ±0.03 (3.1 × 10³) 1.2 × 10⁵ (2.7 × 10³) G89D/R91D RNase 1 R39L/N67L/N88A/0.092 ± 0.003 (1.4 × 10³) 3.1 × 10⁶ (1.1 × 10²) G89L/R91L RNase 1^(a)Values for k_(off) (±SE) were determined by following the release ofa fluorescein-labeled RNase 1 variant from hRI over time and fitting thecurves to eq 1. Values in parentheses represent the fold decrease fromwild-type RNase 1. ^(b)Values of k_(on) were calculated using theequation, K_(d) = k_(off)/k_(on). Numbers in parentheses represent thefold decrease from wild-type RNase 1. ^(c)Value of k_(off) wascalculated using the equation, K_(d) = k_(off)/k_(on). Where the K_(d)value was from Boix et al. and the k_(on) was for hRI/RNase A from Leeet al. ^(d)Value of k_(on) for hRI/RNase A from Lee et al.

The impact of individual mutations in R39D/N67D/N88A/G89D/R91D RNase 1to its overall binding constant is elucidated by the reversion of eachsubstitution in R39D/N67D/N88A/G89D/R91D RNase 1 to the wild-type aminoacid. Residues with little impact on RI affinity will have small ΔΔΔGvalues in Table 5. Small ΔΔΔG values reflect a small change in the ΔΔGvalue when that one residue was reverted to the wild-type amino acid inR39D/N67D/N88A/G89D/R91D RNase 1. The ΔΔΔG values rank the energeticcontributions of the mutations as N88A< G89D< N67D< R39D< R91D.

FIG. 4A plots the binding isotherm of each RNase 1 variant andillustrates the affinity of RNase 1 variants for hRI. Binding to hRI wasdetermined by using a competition assay with fluorescently-labeled G88RRNase A (50 nM). The concentration of bound F*-A19C G88R RNase A wasdetermined by following the decrease in fluorescein emission upon hRIbinding. Data points are the mean (±SE) of at least three separatemeasurements. Variants in order of decreasing hRI-affinity:R39D/N67D/N88A/G89D (▪); N67D/N88A/G89D/R91D (Δ); R39D/N88A/G89D/R91D(●); R39D/N67D/N88A/R91D (◯); R39D/N67D/G89D/R91D (▾);R39D/N67D/N88A/G89D/R91D (␣). The results of this experiment illustratesthis same trend as indicated above for the individual mutations. Anaspartate at position 91 contributed 2.8 kcal/mol of energy to evasion,0.6 kcal/mol more than any other residue. Conversely, aspartates at bothpositions 88 and 89 contributed only 1.5 kcal/mol, which is lower thanthe energetic contribution of any other single substitution, showingthat different residues play a more important role in hRI•RNase 1complex formation as compared to pRI•RNase A.

In regard to R4C/G38R/R39G/N67R/N88L/G89R/R91G/V118C RNase 1, it wasfound that the value of K_(d) for the complex of hRI andR4C/G38R/R39G/N67R/N88L/G89R/R91G/V118C RNase 1 is (5.5±1.6)×10⁻⁸ M. Thevalue of the equilibrium dissociation constant, K_(d), for the complexof hRI with wild-type RNase 1 is not known, but likely to be near thevalue of 10⁻¹⁴ M for the complex of porcine RI with bovine pancreaticribonuclease (RNase A).

Charge and Cytotoxicity

The cytotoxicity of a ribonuclease is governed by all of the precedingribonuclease attributes and the molecular charge of a ribonuclease. Theinterplay between the molecular charge and the cytotoxicity of aribonuclease is seen with the results in FIG. 4B and Table 5.

In particular. FIG. 4B shows the effect of ribonucleases on theproliferation of K-562 cells. The incorporation of [methyl-³H]thymidineinto cellular DNA was used to monitor the proliferation of K-562 cellsin the presence of ribonucleases. Data points are the mean (±SE) of atleast three separate experiments carried out in triplicate. Variants inorder of increasing cytotoxicity: D38R/R39D/N67R/G88R RNase A (Λ); G88RRNase A (◯); R39D/N67D/N88A/G89D/R91D RNase 1 (▴);R39L/N67L/N88A/G89L/R91L RNase 1 (●); N67D/N88A/G89D/R91D RNase 1 (♦);G38R/R39G/N67R/N88R RNase 1 (▪); and wild-type RNase 1 (␣). All othervariants of RNase 1 from Table 5 had curves comparable to that ofG38R/R39G/N67R/N88R RNase 1 and, for clarity, are not shown.

FIG. 4B illustrates that D38R/R39D/N67R/G88R RNase A has the sameconformational stability, a 6-fold higher k_(cat)/K_(M) value, and a3-fold lower K_(d) value than R39D/N67D/N88A/G89D/R91D RNase 1, buttheir IC₅₀ values differ by a disproportionately large 87-fold.R39D/N67D/N88A/G89D/R91D RNase 1 has an IC₅₀ of only 13.3 μM, making itmore toxic than wild-type RNase 1 (FIG. 4), but 2-fold less toxic thanG88R RNase A. The only biochemical characteristic that differssignificantly between D38R/R39D/N67R/G88R RNase A (+6) andR39D/N67D/N88A/G89D/R91D RNase 1 (0) is the net charge.

The IC₅₀ values of all the other variants of RNase 1 from Table 5 falloutside the measurable range of the assay. R39L/N67L/N88A/G89L/R91LRNase 1 and N67D/N88A/G89D/R91D RNase 1, however, have killedapproximately 60% of the K-562 cells at 25 μM (FIG. 4), putting theirIC₅₀ values just slightly above 25 μM. The lack of cytotoxicity for mostRNase 1 variants other than R39D/N67D/N88A/G89D/R91D RNase 1 can beexplained by an increased affinity for RI or loss in thermal stabilitywhen compared to R39D/N67D/N88A/G89D/R91D RNase 1. The outlier to thistrend is N67D/N88A/G89D/R91D RNase 1, whose K_(d) value is 57-fold lowerthan R39D/N67D/N88A/G89D/R91D but whose IC₅₀ value is estimated to beonly 2-fold higher. Part of the anomalous cytotoxicity ofN67D/N88A/G89D/R91D RNase 1 can be attributed to a 3-fold increase inthe catalytic activity over R39D/N67D/N88A/G89D/R91D RNase 1, but theincreased activity does not completely explain the disproportionatelyhigh cytotoxicity of N67D/N88A/G89D/R91D RNase 1.

Furthermore, in assays we conducted with human chronic myelogenousleukemia cell line K-562, wild-type RNase 1 has an IC₅₀ value of >50 μM.In contrast, R4C/G38R/R39G/N67R/N88L/G89R/R91G/V118C RNase 1 exhibitssignificant cytotoxic activity, having an IC₅₀ value of 15 μM.

Taken together, the preceding examples demonstrate the creation ofcytotoxic ribonuclease variants by exploiting the electrostaticinteraction between hRI and RNase 1, such that the variants evade RIbinding, with little compromise to catalytic efficacy. It is to beunderstood that the present invention is not limited to the particularembodiments disclosed in this application, but embraces all suchmodified forms thereof that come within the scope of the followingclaims.

Human Ribonuclease as a Chemotherapeutic

Ribonucleases show great promise as cancer chemotherapeutics.₁₂ ONC, ahomologue of RNase 1 from the northern leopard frog, is currently inphase III clinical trials for the treatment of malignant mesothelioma.However, therapeutics of RNase 1 have multiple advantages over ONC,including enhanced catalytic activity,₁₃ decreased renal toxicity, anddecreased immunogenicity. To develop therapeutics of ribonucleases thatare not naturally cytotoxic, requires the careful consideration ofmultiple biochemical attributes including thermal stability, catalyticactivity, charge, and especially RI evasion.

Variants of RNase 1 with lower affinity for hRI have been difficult toengineer using natural amino acid substitutions. However, using thestructural and electrostatic information obtained from the crystalstructure of the hRI•RNase 1 complex, we have removed this hindrance tocytotoxicity by designing a variant of RNase 1 with an affinity for RIin the micromolar range. R39D/N67D/N88A/G89D/R91D RNase 1 has nearnative catalytic activity and conformational stability, but itscytotoxicity is hindered by lowered positive charge (Table 5 and FIG.4). Ribonuclease variants with lower net charge have increased IC₅₀values when compared to ribonucleases with similar activity, stability,and RI affinity.₁₃ However, the charge on a ribonuclease can beincreased by adding additional positive charge to the termini, whichincreased the toxicity of RNase A. By greatly decreasing the inhibitionof RNase 1 by hRI, we have eliminated a barrier to ribonucleasecytotoxicity and opened the door to human ribonuclease-based therapies.

The tight inhibitory complexes between ribonucleases and RI provide agood model system for studying the influence of electrostatics on theassociation of protein-protein complexes. To this end, we have looked atRNase 1, the structural and sequence homologue of RNase A, whoseinteraction with hRI has been difficult to predict. By determining theX-ray crystal structure of RNase 1 in complex with hRI and studying keyelectrostatic hotspots, we were able to create variants of RNase 1 withmicromolar affinity for hRI. Substitution of key charged residuescreated the greatest change in binding affinity and suggests a new classof anchor residues for protein-protein interactions, electrostaticanchor residues. Mutation of electrostatic anchors like Arg39 or Arg91changes the affinity by influencing the association rate of the complex.We reduced the affinity of hRI for RNase 1 by 10⁷-fold by exploiting theelectrostatic anchors on RNase 1. Accordingly, the variants of RNase 1that evade RI-binding represent a large step in the development ofchemotherapeutics using human ribonucleases.

All publications and patents mentioned in the above specification areherein incorporated by reference as if expressly set forth herein.Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is understood that certain adaptations of theinvention are a matter of routine optimization for those skilled in theart, and can be implemented without departing from the spirit of theinvention, or the scope of the appended claims.

RELATED PUBLICATIONS

-   1. Shaul, Y. & Schreiber, G. Exploring the charge space of    protein-protein association: a proteomic study. Proteins 60, 341-352    (2005).-   2. Rutkoski, T. J., Kurten, E. L., Mitchell, J. C. & Raines, R. T.    Disruption of shapecomplentarity markers to create cytotoxic    variants of ribonuclease A. J. Mol. Biol. 354, 41-54 (2005).-   3. Mitchell, J. C., Kerr. R. & Ten Eyck, L. F. Rapid atomic density    methods for molecular shape characterization. J. Mol. Graph. Model.    19, 325-330 (2001).-   4. Kobe, B. & Deisenhofer, J. A structural basis of the interactions    between leucine-rich repeats and protein ligands. Nature 374,    183-186 (1995).-   5. Lee, J. E. & Raines, R. T. Cytotoxicity of Bovine Seminal    Ribonuclease. Monomer versus Dimer. Biochemistry 44, 15760-15767    (2005).-   6. Leland, P. A., Schultz, L. W., Kim, B.-M. & Raines, R. T.    Ribonuclease A variants with potent cytotoxic activity. Proc. Natl.    Acad. Sci. U.S.A. 98, 10407-10412 (1998).-   7. Leland, P. A., Staniszewski, K. E., Kim, B.-M. & Raines, R. T.    Endowing human pancreatic ribonuclease with toxicity for cancer    cells. J. Biol. Chem. 276, 43095-43102 (2001).-   8 Gaur. D., Swaminathan, S. & Batra, J. K. Interaction of human    pancreatic ribonuclease with human ribonuclease inhibitor. J. Biol.    Chem. 276, 24978-24984 (2001).-   9. Bosch, M. et al. A nuclear localization sequence endows human    pancreatic ribonuclease with cytotoxic activity. Biochemistry 43,    2167-2177 (2004).-   10. Rajamani, D., Thiel, S., Vajda, S. & Camacho, C. J. Anchor    residues in protein-protein interactions. Proc. Natl. Acad. Sci.    U.S.A. 101, 11287-11292 (2004).-   11. Pous, J. et al. Three-dimensional structure of human RNase 1    delta N7 at 1.9 Å resolution. Acta Crystallogr. D Biol. Crystallogr.    57.498-505 (2001).-   12. Pous, J. et al. Three-dimensional structure of a human    pancreatic ribonuclease variant, a step forward in the design of    cytotoxic ribonucleases. J. Mol. Biol. 303, 49-60 (2000).-   13. Boix. E., Wu, Y., Vasandani, V. M., Saxena, S. K., Ardelt, W.,    Ladner, J. & Youle, R. J. (1996). J. Mol. Biol. 257, 992-1007.

1. A method for inhibiting the proliferation of cancer cells, comprisingdelivering to the cells an effective amount of an engineered humanRibonuclease (RNase 1) polypeptide, said polypeptide comprising SEQ IDNO:2 except for one or more amino acid substitutions in the region ofamino acid residues 85 to 94 and one or more amino acid substitutionsselected from the group consisting of substitutions at position 4, 38,39, 67, and 118 of SEQ ID NO:2.
 2. The method of claim 1, wherein saidamino acid substitutions at positions 4, 38, 39, 67 and 118 are R4C,G38R, R39G, N67R, and V118C.
 3. The method of claim 1, wherein saidamino acid substitution in the region of amino acid residues 85 to 94 isN88L.
 4. The method of claim 1, wherein said amino acid substitution inthe region of amino acid residues 85 to 94 is G89R.
 5. The method ofclaim 1, wherein said amino acid substitution in the region of aminoacid residues 85 to 94 is R91G.
 6. A method for inhibiting theproliferation of cancer cells, comprising delivering to the cells aneffective amount of an engineered human Ribonuclease (RNase 1)polypeptide, said polypeptide comprising SEQ ID NO:2 except for one ormore amino acid substitutions in the region of amino acid residues 85 to94 and one or more of the following amino acid substitutions: R4C, G38R,R39G, N67R, or V118C relative to SEQ ID NO:2.
 7. A method for inhibitingthe proliferation of cancer cells, comprising delivering to the cells aneffective amount of an engineered human Ribonuclease (RNase 1)polypeptide, said polypeptide comprising SEQ ID NO:2 except for one ormore amino acid substitutions in the region of amino acid residues 85 to94 and one or more amino acid substitutions at position 4, 38, 39, 67 or118 of SEQ ID NO:2.
 8. The method of claim 7, wherein said amino acidsubstitutions at position 4, 38, 39, 67 or 118 are R4C, G38R, R39G,N67R, and V118C.
 9. The method of claim 7, wherein said amino acidsubstitution in the region amino acid residues 85 to 94 is N88L.
 10. Themethod of claim 7, wherein said amino acid substitution in the region ofamino acid residues 85 to 94 is G89R.
 11. The method of claim 7, whereinsaid amino acid substitution in the region of amino acid residues 85 to94 is R91G.
 12. The method of claim 1, wherein the amino acidsubstitutions are R4C, G38R, R39G, N67R, N88L, G89R, R91G, and V118C.13. The method of claim 6, wherein the amino acid substitutions are R4C,G38R, R39G, N67R, N88L, G89R, R91G, and V118C.
 14. The method of claim7, wherein the amino acid substitutions are R4C, G38R, R39G, N67R, N88L,G89R, R910, and V118C.
 15. A method for inhibiting the proliferation ofcancer cells, comprising delivering to the cells an effective amount ofan engineered human Ribonuclease (RNase 1) polypeptide, said polypeptidecomprising SEQ ID NO:2 except for the following amino acidsubstitutions: R4C, G38R, R39G, N67R, N88L, G89R, R91G, and V118Crelative to SEQ ID NO:2.
 16. The method of claim 1, 6 or 7, wherein theengineered RNase 1 retains ribonucleolytic activity relative to SEQ IDNO:2.
 17. The method of claim 1, 6 or 7, wherein the engineered RNase 1exhibits enhanced cytotoxic activity relative to SEQ ID NO:
 2. 18. Themethod of claim 1, 6 or 7, wherein the engineered RNase 1 exhibits alower binding affinity for hRI than SEQ ID NO:2.
 19. The method of claim1, 6 or 7, wherein the engineered RNase 1 retains ribonucleolyticactivity, exhibits enhanced cytotoxic activity relative to SEQ ID NO: 2,and has a lower binding affinity for hRI than SEQ ID NO:2.
 20. Themethod of claim 1, 6 or 7, wherein the one or more amino acidsubstitutions in SEQ ID NO:2 electrostatically repel and/or stericallyhinder binding of human Ribonuclease Inhibitor (hRI) to the engineeredRNase 1.